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Cotton drought resistance related gene GhDT1 and application thereof

A technology of cotton and genes, applied in the field of biotechnology applications, can solve the problems of few reports on functional characteristics

Active Publication Date: 2020-03-10
NANJING AGRICULTURAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Previous studies have shown that lipid transfer protein genes play an important role in promoting cotton fiber development, but there are few reports on other functional characteristics

Method used

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  • Cotton drought resistance related gene GhDT1 and application thereof
  • Cotton drought resistance related gene GhDT1 and application thereof
  • Cotton drought resistance related gene GhDT1 and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0035] Example 1 Expression analysis of GhDT1 in different tissues of cotton

[0036] Adopt the method for real-time quantitative PCR to detect the tissue distribution of GhDT1 in the upland cotton genetic standard line TM-1 plant known to those skilled in the art, and take the following measures to ensure the reliability of the detection results: digest and extract the GhDT1 with amplification level DNase I There may be a small amount of genomic DNA contamination in the total RNA. In order to detect whether the genomic DNA is eliminated, a part of the total RNA sample digested with DNase I can be taken out for routine PCR reaction. When no amplified band is found, reverse the reaction At the same time, in order to further verify whether the band amplified by real-time quantitative PCR is the target gene, the PCR product was gel-cut and recovered for sequencing. The specific method is: get TM-1 two leaves and one heart stage root, stem, leaf and ovule and fiber different devel...

Embodiment 2

[0043] Example 2 Analysis of the induced expression of GhDT1 under different stresses and hormone treatments

[0044] The real-time quantitative PCR method was used to detect the expression pattern of GhDT1 in the root tissue of upland cotton TM-1 under different treatment conditions, including NaCl, PEG, ABA hormones, etc., respectively, for 0h, 2h, 4h, 6h, 8h, 10h, 12h , 24h.

[0045] Implementation steps, implementation methods and primers used are the same as in Example 1, and the detection results show that GhDT1 is induced by salt, drought and ABA ( figure 2 ).

Embodiment 3

[0046] Example 3 Creation and Identification of Transgenic GhDT1 Gene Cotton Material

[0047] Agrobacterium-mediated genetic transformation method was used to obtain stable transgenic cotton plants, and DNA and mRNA levels were identified according to the promoter sequence, reporter gene and marker gene sequence carried on the vector.

[0048] The specific implementation steps are as follows: Utilize the pCAMBIA2301 plasmid vector purchased in the market to insert GhDT1 between XbaI and Sac I of the pCAMBIA2301 vector, and construct the CaMV35S constitutive expression promoter of the GhDT1 gene by techniques known to those skilled in the art The driven overexpression vector and the antisense interference vector are transformed into cotton through the Agrobacterium-mediated transformation method to obtain stable genetic transgenic lines.

[0049] Identification at the DNA level by PCR methods, including:

[0050] 1. Cotton genomic DNA extraction method

[0051] The leaves of...

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Abstract

The invention discloses a cotton drought resistance related gene GhDT1 and an application thereof, and belongs to the field of biotechnology application. The invention relates to the gene GhDT1 whichencodes a lipid transfer protein. The invention provides a full-length ORF nucleotide sequence and an amino acid sequence of GhDT1 in an allotetraploid upland cotton genetic standard line TM-1. The gene GhDT1 in the cotton undergoes dominant expression in fiber tissues and is subjected to abiotic stress and hormone-induced expression. Overexpressed and interference expressed GhDT1 transgenic cotton materials are obtained through an agrobacterium mediated genetic transformation method. Through identification of natural drought in the seedling stage of a progeny homozygous transgenic line, it isfound that compared with a transgenic receptor material, a plant containing the overexpressed gene has higher drought tolerance, drought tolerance is remarkably reduced through interference expression, and it is indicated that the gene plays an important role in the drought stress resisting process of cotton.

Description

technical field [0001] The invention belongs to the application field of biotechnology, and relates to a cotton GhDT1 gene and its application. The gene encodes a lipid transfer protein. Systematic analysis of members of the Lipid transfer proteins (LTPs) family in cotton revealed that the GhDT1 gene was significantly induced by abiotic stresses, such as salt, drought and hormones. The GhDT1 gene in cotton was predominantly expressed in fiber tissue, and its expression was significantly up-regulated by PEG6000 treatment. The overexpression and interference expression transgenic cotton materials were obtained through the method of Agrobacterium-mediated genetic transformation. The identification of natural drought at the seedling stage of the homozygous transgenic lines in the offspring found that compared with the transgenic recipient material, overexpression of this gene can significantly improve the drought tolerance of plants, and interference expression can significantly ...

Claims

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Application Information

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IPC IPC(8): C12N15/82A01H5/00A01H6/60C12N15/29C07K14/415
CPCC07K14/415C12N15/8273Y02A40/146
Inventor 郭旺珍张大勇李吉宁
Owner NANJING AGRICULTURAL UNIVERSITY
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